Restoration Treatment And Dna Analysis

It is possible to amplify even longer DNA sequences of about 300 bp from postmortem formalin-fixed and paraffin-embedded tissues utilizing a simple restoration treatment. The restoration method is based on the fact that DNA degradation is connected with random single-strand breaks. To obtain longer stretches of DNA, a pre-PCR treatment is performed. It consists of a DNA rehybridiza-tion step in a specific buffer at 55°C[4] followed by a DNA polymerization at 72°C for 20 min. This single-cycle PCR at the elongation temperature (72°C) restores the nicks in the rehybridized DNA using the other corresponding strand as a template.[4] Because of the random position of the nicks, the fragment reconstruction gives sequences long enough for successive PCR analysis. The treated samples are then normally stored at — 20°C until specific PCR analyses. To obtain a PCR amplification with restored DNA in postmortem archive tissues, a denatur-ation step at 95°C is required before PCR amplification, because in the restoration itself the double-stranded DNA is strongly linked with a more stable secondary structure.1-4-1 As reported in Fig. 1A, the PCR amplification in untreated necropsy tissues was unsuccessful, but in reconstructed and denaturated ones the amplification fragments are evident. The fact that the PCR amplification was obtained in formalin-fixed, paraffin-embedded biopsy specimens (Table 1), even without the restoration procedure, confirms that more extensive DNA degradation is confined to postmortem tissues. This simple restoration/denaturation procedure can overcome the problem of DNA degradation in the analysis of necropsy archive tissue. This could eventually result in better suitability of archival DNA for genetic investigations and would overcome the limit for longer-size PCR fragments for specific needs.

Table 1 Polymerase chain reaction amplification of DNA of autopsy and biopsy origin
Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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